The exploration of the antibacterial mechanism of fe3+ against bacteria

MICROBIAL PHYSIOLOGY

The exploration of the antibacterial mechanism of fe³⁺ against bacteria

Hong-qi Sun; Xue-mei Lu; Pei-ji Gao^*

State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China

ABSTRACT

This study demonstrated that the bacteria could adsorb Fe³⁺ and reduce Fe³⁺ to Fe²⁺. Iron had significant bacteriostatic effects, which were directly proportional to the iron concentration and under the influence of pH and chelator. It presumed that the inhibition of Fe³⁺ acts through the formation of hydroxyl free radicals.

Key words: antibacterial activity, chelator, hydroxyl radical, ferric ion, siderophore

Iron is a nutritionally essential trace element and has important functions in the metabolic processes of aerobic organisms, such as photosynthesis, respiration, oxygen transport, gene regulation and DNA biosynthesis (1, 3). Under physiological conditions, iron mainly exists in two readily reversible redox states, the reduced Fe²⁺ form and the oxidized Fe³⁺ form. Although abundant in nature, iron tends to form highly insoluble hydroxides in the aerobic neutral pH environment. The concentration of free ferric ion in solution at biological pH is probably 10^-9-10^-18 mol l^-1 (2, 5, 9), a concentration too low to allow growth by aerobic microorganisms. Microorganisms have evolved a range of strategies to acquire iron. The major strategies include production and utilization of siderophores, utilization of host iron proteins such as transferrin, and lactoferrin, and reduction of Fe³⁺ to Fe²⁺ with subsequent transport of Fe²⁺ (4). On the other hand, iron is a devastating metal. Fe²⁺ reacts with H₂O₂ to form a hydroxyl radical (6). HO· is known to be a highly reactive, indiscriminate oxidizing agent, which can damage proteins and nuclear acids. Thus, aerobic organisms have a dilemma: they need a scavenging system that is effective in accumulating iron, without allowing too much to accumulate. Much attention has been given to the response of bacteria to iron-limited conditions, but not to how the bacteria respond to adequate or excess iron supply. The growth of bacteria usually requires 10^-6 mol l^-1 of iron, and the proper amount of iron can stimulate the growth of bacteria, but under excessive concentrations, the microorganism's growth will be inhibited.

In this article, we studied the adsorption and reduction of 2 Gram-negative bacteria (Escherichia coli CVCC 249 and Ralstonia solanacearum) and 2 Gram-positive bacteria (Staphylococcus aureus ATCC 25923 and Bacillus subtilis) to Fe³⁺ under excess Fe³⁺ conditions. The antibacterial effects of Fe³⁺ were investigated and we conducted a preliminary analysis of the inhibition mechanism of Fe³⁺.

Each bacterial strain was inoculated into 100 ml LB broth and incubated at 37ºC with shaking at 170 rpm for 12 h. The culture was centrifuged and washed twice with sterile physiological saline, then the pellet was resuspended in appropriate amount of sterile physiological saline and the turbidity of the samples was measured at 600 nm using a UNICO 2000 UV-Vis spectrophotometer, made the turbidity values were 2.0. A sample volume of 5 ml of bacteria suspension was centrifuged and the cell was resuspended into 5 ml of FeCl₃ solution (1 mmol l^-1, containing 2.5 mmol l^-1 HCl) and then incubated at 37ºC, with shaking at 170 rpm for 2 h. Similarly, the 5 ml bacteria suspension containing 5 ml FeCl₃ solution (1, 0.5 and 0.25 mmol l^-1, containing 2.5 mmol l^-1 HCl, respectively) was incubated at room temperature with shaking for 0-45 min at every 5 min interval and then it was centrifuged the supernatant was collected. The ferrous and ferric ion concentrations in the supernatant were measured using 1, 10-phenanthroline spectrophotometry at 510 nm and 364 nm, respectively (8, 12). A volume of 1 ml hydroxylamine hydrochloride (w/v 5%) was added to 1 ml of supernatant, incubated 15 min, and then mixed with 1 ml 1, 10phenanthroline (m/v, 0.25%), and 1 ml sodium acetate buffer (0.5 mol l^-1, pH 4.5), and then added to deionized water to make a total volume of 5 ml. The mixture was further incubated at room temperature for about 30 min, after which the absorbance at 510 nm was measured to determine the total iron content in the supernatant. The hydroxylamine hydrochloride was replaced with the same amount of deionized water and the absorbance at 510 nm and 364 nm were measured to characterize the Fe²⁺ and Fe³⁺ ion content, respectively. A solution of 1 mmol l^-1 FeCl₃ was used as a control. The results showed that all four bacteria could adsorb Fe³⁺ . R. solanacearum had the strongest adsorptive capacity for Fe³⁺ at about 50%, followed by B. subtilis and S. aureus ATCC 25923 which retained about 30% while the absorption capacity of E. coli CVCC 249 for Fe³⁺ was the weakest at close to 15%. As can be seen from Figure 1, the iron absorption of the 4 bacteria reached equilibrium in 5-10 min and thus, the adsorption was an extremely rapid process. The four bacteria had similar absorption dynamics curves of total iron and Fe³⁺ . For E. coli CVCC 249, S. aureus ATCC 25923 and B. subtilis bacteria, the Fe³⁺ adsorption could achieve saturation when the Fe³⁺ concentration was 0.25 mmol l^-1, but for R. solanacearum, saturation could only be reached when the concentration increased to 0.5 mmol l^-1. This indicated that R. solanacearum had the strongest Fe³⁺ adsorptive capacity. The results also indicated that the decrease of the total iron and Fe³⁺ content was accompanied by the generation of Fe²⁺ . The dynamics curve of the Fe²⁺ production was similar for the 4 bacteria. Moreover, the Fe²⁺ production of R. solanacearum and B. subtilis was higher than E. coli CVCC 249 and S. aureus ATCC 25923, which corresponded directly with their Fe³⁺ adsorptive capacity.

To investigate the antibacterial effects of high concentrations of ferric iron, the appropriate amount of FeCl₃ was sterilized using ultraviolet radiation and then it was combined with sodium hydrogen phosphate -citric acid buffer solutions (pH 4.0, 5.0, 6.0, and 7.0, prepared using 10 mmol l^-1 sodium hydrogen phosphate and 5 mmol l^-1 citric acid, separately and then sterilized at 121ºC for 30 min.) or sterile water to compound the FeCl₃ solutions separately. It was prepared just before use and diluted to the necessary concentrations. The bacterial culture was appropriately diluted with sterile saline and then mixed with different concentrations and pHs of FeCl₃ solution at room temperature for 10, 20, 30, and 40 min, respectively. A volume of 100 µl of the solution was spread onto a LB plate. Plates were incubated at 37ºC overnight, and colonies on the plates were counted. Samples were plated in triplicate and sterile saline was used as a negative control. The results illustrates that the different concentrations of FeCl₃ aqueous solutions (without buffer) had definite antibacterial activities. The bactericidal capacity of Fe³⁺ increased with the increasing concentration of Fe³⁺ . Additionally, the inhibitory effect was an extremely rapid process. In 10 min, 70% of E. coli CVCC 249 bacteria were inhibited by 1 mmol l^-1 Fe³⁺, and 90% of S. aureus ATCC 25923 were inhibited at the same concentration. Both R. solanacearum and B. subtilis were completely inhibited when the Fe³⁺ concentration was only 250 µmol l^-1 and 62.5 µmol l^-1 , respectively. The antibacterial results of four pH Fe³⁺ solutions on the growth of the bacteria. The four different pH buffer solutions did not obviously inhibited E. coli CVCC 249 and S. aureus ATCC 25923. However, for R. solanacearum and B. subtilis, they had varying degrees of inhibition while a solution with a pH value of 4.0 could suppress their growth completely. For solutions with pH values of 4.0, 5.0, and 6.0, 1 mmol l^-1 Fe³⁺ could suppress the growth of E. coli CVCC 249, while at 0.5 mmol l^-1 Fe³⁺ no inhibitory effects were observed. For the pH 7.0 solution, only the 2 mmol l^-1 Fe³⁺ displayed inhibitory effects. Under all pH conditions, only 2 mmol l^-1 Fe³⁺ could obviously inhibit the growth of S. aureus ATCC 25923. For solutions with pH values of 5.0 and 6.0, 500 µmol l^-1 and 250 µmol l^-1 Fe³⁺ had obvious inhibitory effects on the growth of R. solanacearum and the B. subtilis, respectively. When the pH value was 7.0, the inhibitory effects weakened. By comparison of these results, it was determined that the antibacterial effects of the buffered Fe³⁺ solutions were weaker than those of the Fe³⁺ aqueous solutions. Thus, it is necessary that higher concentrations of FeCl₃ were needed in buffered solutions in order to achieve the same antibacterial efficiency. During the preparation of the fresh FeCl₃ solution, the hydrolysis state of ferric ion changes continuously. Depending on the pH of the sodium hydrogen phosphate -citric acid buffer solution, the Fe³⁺ will be in different hydrolysis forms which have correspondingly different relative antibacterial capacities. At pH 7, the concentration of free ferric ion is 1.4 × 10^-9 mol l^-1 in the aqueous solution and the main species is Fe(OH)₂⁺. In the presence of 40 mmol l^-1 phosphate, the concentration of free ferric ion is decreased to 3.3 × 10^-12 mol l^-1 because of the formation of ferric phosphate (Chipperfield and Ratledge 2000). Depending on the different pH conditions, the hydrolysis of Fe³⁺ and the formation of ferric phosphate complexes results in a decline in the free Fe³⁺ content, which make the antibacterial activity of the iron weaker. The sensitivities of the various bacteria to the ferric iron were different, but the bacteriostasis dynamics curves were the same. This indicates that the antibacterial effect of Fe³⁺ is likely a non-selective mechanism.

The FeCl₃ solution was mixed with three different chelator solutions, acetohydroxamic acid (ICN Biomedicals), 2, 3-dihydroxybenzoic acid (Acros Organics) or a siderophore which produced by Aspergillus niger An76 (11) for 2 min, and then added into a bacterial suspension at room temperature for 10 min, respectively. In addition, the FeCl₃ solution was added into the bacterial suspension at room temperature for 5 min and then mixed with the above three chelator solutions for 5 min, respectively. Results of the comparison between the antibacterial activity of Fe³⁺ and chelators are shown in Figure 2. Solutions of Fe³⁺ alone displayed remarkable bacteriostasis. The inhibitory rates of 1 mmol l^-1 Fe³⁺ for E. coli CVCC 249 and S. aureus ATCC 25923 were approximate 80% within a 10 min treatment time, and 83 µmol l^-1 Fe³⁺ could completely suppress the growth of R. solanacearum and B. subtilis. All chelators had no obvious inhibitory effects on E. coli CVCC 249, but did inhibit S. aureus ATCC 25923. 2, 3-Dihydroxybenzoic acid could strongly inhibit the growth of B. subtilis. Acetohydroxamic acid showed a certain extent inhibitory effect for S. aureus ATCC 25923, but for other three bacteria did not obvious inhibitory action. Aspergillus niger An76 siderophore can completely inhibit R. solanacearum and B. subtilis growth. When 2, 3-dihydroxybenzoic acid or acetohydroxamic acid was combined with Fe³⁺ together, it was determined that both of the chelators could enhance the Fe³⁺ inhibition of E. coli CVCC 249 and S. aureus ATCC 25923 regardless of whether they were added before or after the Fe³⁺ solutions were mixed with the bacterial suspension. But the An76 siderophore could reduce the Fe³⁺ inhibition of E. coli CVCC 249 and S. aureus ATCC 25923.

Acetohydroxamic acid and 2, 3-dihydroxybenzoic acid can form complexes with Fe³⁺, which cause the hydrolysis of ferric ion to be suppressed. One molecule of Fe³⁺ can bind to the 2, 3-dihydroxybenzoic acid between the acid group and a neighboring hydroxyl group and form an iron complex that subsequently reduces an additional Fe³⁺ (13). The 2, 3-dihydroxybenzoic acid - Fe³⁺ complex can catalyze the generation of hydroxyl free radicals which reach a maximum at about a 1:1 ratio (10). The Fenton reagent, which is a complex of hydrogen peroxide and an iron catalyst, has a strong sterilization action, which has been proven to work through HO·. In our study, 4 bacteria were able to rapidly reduce Fe³⁺ to Fe²⁺. Moreover, H₂O₂ is a normal metabolite in aerobic and facultative aerobic organisms (7). Our previous research demonstrated that the siderohpre produced by Aspergillus niger An76 has significantly scavenging activity of hydroxyl free radicals (11). Therefore, it is presumed that pure Fe³⁺ does not have the antibacterial effect alone, but instead, it becomes effective as an inhibitor after the first reduction of Fe³⁺ to Fe²⁺ , and further oxidation with oxidized factors, such as superoxide radical and hydrogen peroxide, with the ultimate formation of hydroxyl free radicals which have a strong non-selective sterilization ability.

The formation of drug resistant microorganisms remains a difficult medical problem. Non-selective sterilization would be a useful technique to combat drug resistance and the information provided herein should provide knowledge towards making a disinfectant using iron chelators and iron. However, the interaction of iron ions with chelating agents and their inhibitory mechanism still needs more in-depth research.

ACKNOWLEDGEMENTS

This work was supported by the Natural Science Foundation of Shandong Province (Grant No. Y2004D07).

Submitted: June 06, 2009; Approved: June 21, 2010.

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Publication Dates

• Publication in this collection
  10 Jan 2011
• Date of issue
  Mar 2011

History

• Received
  06 June 2009
• Accepted
  21 June 2010